Methods for transfecting T cells

ABSTRACT

A method for transfecting T cells with a nucleic acid molecule comprising a gene such that the gene is expressed in the T cells is described. The T cells are stimulated and proliferating prior to introduction of the nucleic acid molecule.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.08/435,095 now abandoned, filed May 4, 1195, entitled “Methods forModulating Expression of Exogenous DNA in T Cells”, the entire contentsof which are incorporated herein by reference.

GOVERMENT SUPPORT

Work described herein was supported in part by NMRDC grant 61153NAE.4120.001.1402. The U.S. government therefore may have certain rightsin the invention.

BACKGROUND

The expression of exogenous DNA in eukaryotic cells permits the study ofa broad array of biological topics ranging from the regulation of geneexpression to the treatment of disease by gene transfer-based therapies.A number of methods for gene transfer into mammalian cells have evolved.These include in vivo and in vitro infection with cloned retroviralvectors (Shimotohno, K., and Temin, H. M. (1981) Cell 26:67-77; Cone, R.D., and Mulligan, R. C. (1984) Proc. Natl. Acad. Sci. USA 81:6349-6353;Dubensky, T. W., Campbell, B. A., and Villareal, L. P. (1984) Proc.Natl. Acad. Sci. USA 81:7529-7533; Seeger, C., Ganem, D. and Varmus, H.E. (1984) Proc. Natl. Acad. Sci. USA 81:5849-5852), coprecipitation ofDNA with calcium phosphate (Chu, G., and Sharp, P. (1981) Gene13:197-202; Benvenisty, N., and Reshef, L. (1986) Proc. Natl. Acad. Sci.USA 83:9551-9555), encapsulation of DNA in liposomes (Felgner, P. L.,and Ringold, G. M. (1989) Nature 337:387-388; Kaneda, Y., Iwai, K., andUchida, T. (1989) Science 243:375-378), direct injection of plasmid DNA(Wolff, J. A., Malone, R. W., Williams, P., Chong, W., Acsadi, G., Jani,A., and Felgner, P. L. (1990) Science 247:1465-1468), DEAE-dextran(McCutchan, J. H., and Pagano, J. S. (1968) J. Natl. Cancer Inst.41:351-357), electroporation (Neumann, E., Schaefer-Ridder, M., Wang,Y., and Hofschneider, P. H. (1982) EMBO J. 1:841-845; Cann, A. J.,Koyanagi, Y., and Chen, I. S. Y. (1988) Oncogene 3:123-128), andDNA-coated particle bombardment of cells and tissues (Yang, N-S.,Burkholder, J., Roberts, B., Martinell, B., and McCabe, D. (1990) Proc.Natl. Acad. Sci. USA 87:9568-9572).

Although transfection of numerous cell types with an exogenous nucleicacid molecule containing a gene results in efficient expression of theexogenous gene, primary T lymphocytes, e.g. peripheral blood Tlymphocytes obtained from an individual, have been found to berefractory to transfection and expression of exogenous DNA. Primary Tlymphocytes also have been found to be refractory to expression of theintroduced nucleic acid when first stimulated to proliferate. Thus, asystem that allows for efficient introduction of exogenous DNA intoprimary T cells and expression of the exogenous DNA in the T cell isstill needed.

SUMMARY

The present invention provides an improved method for transfecting Tcells with a nucleic acid molecule containing a gene such thatexpression of the gene in the T cells is enhanced as compared to classictransfection techniques. The method of the invention is particularlyuseful for transfecting primary T cells which are refractory toclassical transfection techniques. The method involves contacting aproliferating T cell with one or more agents which stimulate theproliferating T cell prior to introducing the nucleic acid molecule intothe T cell. In one embodiment of the invention, the T cell is stimulatedwith a combination of a first agent which provides a primary activationsignal to the T cell and a second agent which provides a costimulatorysignal. The method of the invention has numerous applications, inparticular for gene therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents graphically the relative cell number and cell volumeof CD28⁺ T cells at day 1, 5, 7, 9, and 11 following stimulation (at day0) with anti-CD3 coated plates and anti-CD28 at 1 μg/ml.

FIG. 2 represents a growth curve of freshly isolated CD28⁺ T cellsstimulated with a saturating quantity of immobilized anti-CD3 mAb G19-4(αCD3) in the presence of the anti-CD28 mAb 9.3 (αCD28).

FIG. 3 is a Northern blot indicating the levels of Ets-1 (ETS-1) andhuman leukocyte antigen (HLA) mRNA in primary CD28⁺ T cells cultured for0, 6, 24, and 72 hours after the first stimulation (day 1) or a secondstimulation (day 8) with a saturating quantity of immobilized anti-CD3mAb G19-4 (αCD3) and anti-CD28 mab 9.3 (αCD28).

FIG. 4 is a schematic representation of an example of a transfectionprotocol, in which resting T cells (Rest) are first incubated withanti-CD3 and anti-CD28 (αCD3+αCD28) for two days, followed by incubationwith anti-CD28 alone (αCD28) for 3 days, stimulated (primarystimulation) 10 hours prior to transfection, transfected at T=0,restimulated (secondary stimulation) at 30 hours post transfection, andharvested 10 hours later.

FIG. 5 represents the results of CAT assays performed with cell extractsfrom exponentially growing T cells transfected with RSV-CAT stimulated10 hours before transfection with phorbol-12,13-dibutyrate and ionomycin(PDBU+IONO) or conditioned medium alone (MED) and harvested 40 hourspost transfection. Normalized CAT activity is expressed as (%acetylation/mg protein)×50.

FIG. 6 (Panels A-D) depicts the results of flow cytometric analysis ofacridine orange stained primary T cells and proliferation assays ofprimary T cells cultured under the following conditions: untreatedresting primary T cells (Panel A), T cells stimulated for 3 days withanti-CD3 and anti-CD28 (Panel B), T cells stimulated for 3 days withanti-CD3 and anti-CD28 and then incubated in fresh medium for another 3days (Panel C), or T cells stimulated for 3 days with anti-CD3 andanti-CD28, stimulated for 10 hours with phorbol-12,13-dibutyrate (PDBU)plus ionomycin and then incubated in fresh medium for another 2 days and14 hours (panel D). The graphic representations of flow cytometricanalysis of acridine orange stained cells indicate the DNA and RNAcontent of the cells, which is indicative of the number of cells in G0(%G0), G1 (%G1), and S/G2M (%S/G2M) phase of the cell cycle.

FIG. 7 represents the results of CAT assays performed with cell extractsfrom exponentially growing T cells transfected with RSV-CAT andstimulated 10 hours before transfection (1°) with medium alone (MED) orphorbol-12,13-dibutyrate and ionomycin (PDBU+IONO) and stimulated 30hours after transfection (2°) with medium alone (MED),phorbol-12,13-dibutyrate and ionomycin (PDBU+IONO), anti-CD3 andanti-CD28 antibodies (αCD3+αCD28), or conditioned medium (COND MED) andharvested 10 hours later. Normalized CAT activity is expressed as (%acetylation/mg protein)×50.

FIG. 8 represents the results of CAT assays performed with cell extractsfrom exponentially growing T cells transfected with an HIV-1-CATexpression construct and stimulated 10 hours before transfection (1°)with media alone (MED), phorbol-12,13-dibutyrate and ionomycin(PDBU+IONO), or anti-CD3 and anti-CD28 antibodies (αCD3+αCD28) andstimulated 30 hours after transfection (2°) with media alone (MED),phorbol-12,13-dibutyrate and ionomycin (PDBU+IONO), anti-CD3 andanti-CD28 antibodies (αCD3+αCD28), or conditioned medium (COND MED) andharvested 10 hours later. Normalized CAT activity is expressed as (%acetylation/mg protein)×50.

FIG. 9 represents total RNA content (Panel A) or levels of mRNA for IL-2(Panel B) and HLA (Panel C) determined by Northern blot analysis inCD28⁺ T cells cultured with medium alone (MED) for 12 hours or withanti-CD3 antibody (CD3) for 1, 6, 12, and 24 hours. Panel D indicatesthe amount of IL-2 produced by the T cells incubated with medium alone(MED) for 12 hours or with anti-CD3 antibody (CD3) for 1, 6, 12, 24, or48 hours and the percentage of the cells in phase S, G2 or M of the cellcycle after 48 hours of culture with anti-CD3.

FIG. 10 represents the results of CAT assays performed with cellextracts from exponentially growing T cells transfected with an IL2-CATexpression construct and stimulated 10 hours before transfection (1°)with media alone (MED) or phorbol-12,13-dibutyrate and ionomycin(PDBU+IONO) and stimulated 30 hours after transfection (2°) with mediaalone (MED), phorbol-12,13-dibutyrate and ionomycin (PDBU+IONO),anti-CD3 and anti-CD28 antibodies (αCD3+αCD28), or conditioned medium(COND MED) and harvested 10 hours later. Normalized CAT activity isexpressed as (% acetylation/mg protein)×50.

FIG. 11 represents the results of CAT assays performed with cellextracts from resting T cells transfected with RSV-CAT and treated 10hours before transfection with medium alone (Day 1: MED), anti-CD28 (Day1: αCD28), Staphylococcal enterotoxin A (Day 1: SEA), orphorbol-12,13-dibutyrate and ionomycin (Day 1: PDBU+IONO), or withanti-CD3 and anti-CD28 for 5 days and then for 10 hours beforetransfection with conditioned medium (Day 6: MED) orphorbol-12,13-dibutyrate and ionomycin (Day 6: PDBU+IONO). NormalizedCAT activity is expressed as (% acetylation/mg protein)×50.

FIG. 12 shows autoradiograms of Southern blots of nuclear DNA (NUCLEAR,Panel A) or cytoplasmic DNA (CYTO, Panel B) extracted from proliferatingT cells 0, 6, 24, or 48 hours after transfection of the proliferating Tcells with RSV-CAT or no plasmid (MOCK), stimulated 10 hours prior tothe transfection with phorbol-12,13-dibutyrate and ionomycin (PDBU+IONO)or conditioned medium alone (MED) and hybridized with an EcoRI fragmentfrom the CAT coding region of RSV-CAT. Plasmid DNA corresponding to 1(1c), 10 (10c), 100 (100c), and 1000 (1000c) copies of RSV-CAT/cell wasused as a control. (lin) linear plasmid; (sc) supercoiled plasmid. Thesize of fragments (in kilobases, kb) from a molecular weight marker isrepresented on the left of the Southern blots.

FIG. 13 shows autoradiograms of Southern blots of nuclear DNA extractedfrom exponentially growing T cells transfected with RSV-CAT andstimulated 10 hours before transfection (1°) with medium alone (MED) orphorbol-12,13-dibutyrate and ionomycin (PDBU+IONO) and stimulated 30hours after transfection (2°) with medium alone (MED),phorbol-12,13-dibutyrate and ionomycin (PDBU+IONO), anti-CD3 andanti-CD28 antibodies (αCD3+αCD28), or conditioned medium (COND MED) andhybridized with an EcoRI fragment from the CAT coding region of RSV-CAT.Plasmid DNA corresponding to 1 (1c), 10 (10c), 100 (100c), and 1000(1000c) copies of RSV-CAT/cell was used as a control. (lin) linearplasmid; (sc) supercoiled plasmid. M: molecular weight marker.

FIG. 14 shows autoradiograms of Southern blots of nuclear DNA extractedfrom exponentially growing T cells transfected with an IL2-CATexpression construct and stimulated 10 hours before transfection (1°)with media alone (MED) or phorbol-12,13-dibutyrate and ionomycin(PDBU+IONO) and stimulated 30 hours after transfection (2°) with mediaalone (MED), phorbol-12,13-dibutyrate and ionomycin (PDBU+IONO),anti-CD3 and anti-CD28 antibodies (αCD3+αCD28), or conditioned medium(COND MED) and hybridized with an EcoRI/BamHI fragment from the CATcoding region of IL2-CAT. Plasmid DNA corresponding to 1, 10, 100, and1000 copies of IL2-CAT/cell was used as a control. (lin) linear plasmid;(sc) supercoiled plasmid. M: molecular weight marker.

FIG. 15 represents the percentage of counts per minute (cpm) recoveredfrom the nuclei (Nuclear) or cytoplasm (Cytoplasmic) of T cellstransfected with ³²P-radiolabeled linearized RSV-CAT and stimulated 10hours before transfection with phorbol-12,13-dibutyrate and ionomycin(PDBU/IONO) or conditioned medium alone (MED) and harvested immediately(0), 6, 24, or 48 hours following the transfection. The percentage ofcounts per minute is calculated relative to the total number of countsper minute added to the cells for transfection.

FIG. 16 represents the results of CAT assays performed with cellextracts from proliferating T cells transfected with RSV-CAT orHIV-1-CAT and treated with medium alone (MED), monocytes (MONO),Staphylococcal enterotoxin A (SEA), or moncytes and Staphylococcalenterotoxin A (MONO+SEA) for 10 hours before transfection and harvested40 hours post transfection. Normalized CAT activity is expressed as (%acetylation/mg protein)×50.

DETAILED DESCRIPTION

The present invention provides an improved method for transfecting a Tcell with a nucleic acid molecule comprising a gene such that the geneis expressed in the T cell. The method of the invention comprisescontacting a proliferating T cell with at least one agent whichstimulates the proliferating T cell prior to introducing the nucleicacid molecule into the T cell, such that the gene is expressed in the Tcell.

The method of the invention is based, at least in part, on theobservation that transfection of primary T cells with a nucleic acidcomprising a gene results in poor expression of the gene unless theprimary T cells are proliferating and, furthermore, are stimulated withstimulatory agents, such as agents which induce a primary activationsignal and a costimulatory signal in the T cells. The T cells arepreferably contacted with the stimulatory agents about 10 hours prior tointroducing the nucleic acid into the T cells. Thus, significantexpression of an exogenous gene can be achieved in T cells bystimulating proliferating T cells prior to introducing a nucleic acidcomprising the gene into the T cells.

Thus, the invention provides an improved method for transfecting T cellswith a nucleic acid molecule comprising a gene such that the gene isexpressed in the T cells. The improvement provided by the methods of theinvention over classical T cell transfection methods involves contactingthe T cell with a stimulatory agent prior to (eg. several hours)introducing the nucleic acid molecule into proliferating T cells. Themethod of the invention allows for much higher expression of the geneintroduced into the T cells than conventional transfection techniques.The method of the invention is particularly useful for introducing andexpressing a gene of interest into primary T cells. Thus, in a specificembodiment of the method, T cells are obtained from a subject,transfected in vitro with a nucleic acid molecule according to themethods of the invention, and readminstered to the host. The gene ofinterest can be a gene encoding a protein, or a gene encoding afunctional RNA molecule, such as an antisense molecule or a ribozyme.The gene of interest can encode any protein of interest, includingproteins that protect the T cells, proteins that are toxic to the Tcells, or proteins that are secreted from the T cells to effect othercells. Thus, the method of the invention is applicable to, for example,gene therapy, alteration of T cell function and production of proteinsin T cells.

1. T Cells That can be Transfected According to the Method of theInvention

The invention involves a method for transfecting a T cell with a nucleicacid molecule comprising a gene, such that the gene is expressed in theT cell. The term “T cell” refers to T lymphocytes as defined in the artand is intended to include thymocytes, immature T lymphocytes, mature Tlymphocytes, resting T lymphocytes, or activated T lymphocytes. The Tcells can be CD4⁺ T cells, CD8⁺ T cells, CD4⁺CD8⁺ T cells, or CD4⁻CD8⁻ Tcells. The T cells can also be T helper cells, such as T helper 1 (Th1)or T helper 2 (Th2) cells. T cells that differ from each other by atleast one marker, such as CD4, are referred to herein as “subsets” of Tcells.

The T cells can be a purified population of T cells, or alternativelythe T cells can be in a population with cells of a different type, suchas B cells and/or other peripheral blood cells. The T cells can be apurified population of a subset of T cells, such as CD4⁺ T cells, orthey can be a population of T cells comprising different subsets of Tcells. In another embodiment of the invention, the T cells are T cellclones that have been maintained in culture for extended periods oftime. T cell clones can be transformed to different degrees. In aspecific embodiment, the T cells are a T cell clone that proliferatesindefinitely in culture.

In a preferred embodiment of the invention, the T cells are primary Tcells. The language “primary T cells” is intended to include T cellsobtained from an individual, as opposed to T cells that have beenmaintained in culture for extended periods of time. Thus, primary Tcells are preferably peripheral blood T cells obtained from a subject. Apopulation of primary T cells can be composed of mostly one subset of Tcells. Alternatively, the population of primary T cells can be composedof different subsets of T cells.

The T cells can be from a healthy individual, or alternatively the Tcells may be from an individual affected with a condition. The conditioncan be an infectious disease, such as a condition resulting from a viralinfection, a bacterial infection or an infection by any othermicroorganism. In a specific embodiment, the T cells are from anindividual infected with a human immunodeficiency virus (HIV). In yetanother embodiment of the invention, the T cells are from a subjectsuffering from or susceptible to an autoimmune disease. The T cells canbe of human origin, murine origin or any other mammalian species.

According to the method of the invention, the nucleic acid molecule isintroduced into T cells that are actively proliferating. T cells can bestimulated to proliferate by contacting the T cells with a variety ofagents, such as a combination of agents providing a primary activationsignal and a costimulatory signal to T cells. Agents that can be used tostimulate T cells to proliferate are well known in the art and aredescribed below, in Section 2. T cells that are stimulated toproliferate are characterized by cellular enlargement, clumping, andacidification of the culture medium. Thus, T cell proliferation can beevidenced by, for example, examining the size or measuring the volume ofthe T cells, such as with a Coulter Counter. A resting T cell has a meandiameter of about 6.8 microns. Following the initial activation andstimulation the T cell mean diameter will increase to over 12 microns byday 4 and begin to decrease by about day 6. Moreover, T cellproliferation can be assessed by standard techniques known in the art,such as tritiated thymidine uptake.

2. Stimulatory Agents

The method of the invention involves contacting proliferating T cellswith at least one stimulatory agent prior to introducing the nucleicacid molecule into the proliferating T cell. The term “stimulatoryagent” is intended to include agents which provide a signal to the Tcell, such that the level of expression of the gene comprised in thenucleic acid molecule transfected into the T cell is higher when the Tcell is contacted with the stimulatory agent prior to introducing thenucleic acid molecule into the T cell, than in T cells not contactedwith the stimulatory agent prior to introducing the nucleic acidmolecule.

In a specific embodiment of the invention, the stimulatory agent is anagent which provides a primary activation signal to a T cell. Thelanguage “primary activation signal” is intended to include signals ,typically triggered through the TCR/CD3 complex, that induce activationof T cells. Activation of a T cell is intended to include modificationsof a T cell, such that the T cell is induced to proliferate anddifferentiate upon receiving a second signal, such as a costimulatorysignal. In a specific embodiment, the primary activation signal isprovided by an agent which contacts the T cell receptor or the CD3complex associated with the T cell receptor. In a preferred embodiment,the agent is an antibody reactive against CD3, such as the monoclonalantibody OKT3 (available from the American Type Culture Collection,Rockville, Md.; No. CRL 8001). In another embodiment of the invention,the stimulating agent is an agent that stimulates the CD2 complex on Tcells, such as a combination of antibodies, e.g. T11.3+T11.1 orT11.3+T11.2 (see e.g., Meuer, S. C. et al. (1984) Cell 3:897-906).

In a preferred embodiment of the invention, the T cells are stimulatedwith a combination of agents that stimulate both a primary activationsignal and a costinulatory signal in the T cell. The term “costimulatoryagent” is intended to include agents which provide a costimulatorysignal in T cells, such that a T cell that has received a primaryactivation signal (e.g. an activated T cell) is stimulated toproliferate or to secrete cytokines, such as IL-2, IL-4, orinterferon-γ. In a specific embodiment, the costimulatory agentinteracts with CD28 or CTLA4 molecules on the surface of the T cells. Inan even more specific embodiment, the costimulatory signal is a ligandof CD28 or CTLA4, such as a B-lymphocyte antigen B7-1 or B7-2. Thelanguage “stimulatory form of a natural ligand of CD28” is intended toinclude B7-1 and B7-2 molecules, fragments thereof, or modificationsthereof, which are capable of providing costimulatory signals to the Tcells. Stimulatory forms of natural ligands of CD28 can be identifiedby, for example, contacting activated peripheral blood lymphocytes witha form of a natural ligand of CD28 and performing a standard T cellproliferation assay. Thus, a stimulatory form of a natural ligand ofCD28 is capable of stimulating proliferation of the T cells. Stimulatoryforms of natural ligands of CD28/CTLA4 are described, for example, inPCT Publication No. WO 95/03408.

Other agents that can be used to stimulate T cells prior to introducinga nucleic acid molecule into the T cell include agents that stimulateone or more intracellular signal transduction pathways involved in Tcell activation and/or costimulation. In a preferred embodiment of theinvention, the stimulatory agent is a calcium ionophore, such asionomycin or A23187. Alternatively, the stimulatory agent can be anagent which stimulates protein kinase C, such as a phorbol ester. Apreferred phorbol ester is phorbol-12,13-dibutyrate. In an even morepreferred embodiment of the invention, T cells are contacted with acombination of a calcium ionophore and a phorbol ester prior totransfection with a nucleic acid molecule. The stimulatory agent canalso be an agent which activates protein tyrosine kinases. A preferredagent that stimulates protein tyrosine kinases is pervanadate (O'Shea,J. J., et al. (1992) Proc. Natl. Acad. Sci. USA 89:10306).

In yet another embodiment of the invention, the stimulatory agent is apolyclonal activator. Polyclonal activators include agents that bind toglycoproteins expressed on the plasma membrane of T cells and includelectins, such as phytohemaglutinin (PHA), concanavalin (Con A) andpokeweed mitogen (PWM).

By providing a clone a specific activation signal, it is possible toselectively transfect only a certain clone of T cells in a population ofT cells. Specific activation of a T cell clone can be accomplished, forexample, using a specific antigen presented by an antigen-presentingcell.

In yet another embodiment of the method, the stimulatory agent is alymphokine, such as IL-2. The lymphokine is preferably used incombination with another agent, such as an agent which provides aprimary activation signal to the T cell, for stimulating T cells. Thus,in a preferred embodiment of the invention, T cells are contacted with acombination of an agent which provides a primary activation signal tothe T cells (e.g., an anti-CD3 antibody) and an effective amount ofIL-2, prior to transfecting the T cells with a nucleic acid molecule,such that the nucleic acid molecule is expressed in the T cells.

Other stimulating agents that can be used include super-antigens. Theterm “super-antigen” as defined herein is intended to include bacterialenterotoxins, or other bacterial proteins capable of stimulatingproliferation of T cells. Super-antigens include staphylococcalenterotoxins (SE), such as SEA, SEB, SEC, SED, and SEE. Super-antigenscan also be of viral origin, such as retroviral super-antigens.

Additional agents that are capable of stimulating T cells, either aloneor in combination with other agents, that may be identified using T cellstimulation assays as known in the art or described herein are alsowithin the scope of the invention. For stimulating T cells prior tointroduction of a nucleic acid molecule into the T cells, anycombination of the above described agents can be used.

The stimulating agents can be used in solution, or attached to a solidsurface. The solid surface can be, for example, the surface of a tissueculture dish or a bead. Depending on the nature of the stimulatoryagent, linkage to the solid surface can be performed by methods wellknown in the art. For example, proteins can be chemically crosslinked tothe cell surface using commercially available crosslinking reagents(Pierce, Rockford Ill.) or immobilized on plastic by overnightincubation at 4° C. If several agents are used for stimulation of the Tcells, some agents may be in solution and some agents may be attached toa solid support. In a preferred embodiment, the T cells are stimulatedwith a combination of solid phase coupled anti-CD3 antibody and solubleanti-CD28 antibody.

The specific doses of stimulatory agent(s) to be added to the T cellswill vary with the type of stimulating agent. Typically, the stimulatingagents are used at the same doses at which they are used for stimuling Tcells to proliferate and secrete cytokines, as described in the art.

3. Protocols for Stimulation Prior to Transfection

In a specific embodiment of the invention, T cells are contacted withthe stimulatory agent prior to introducing the nucleic acid moleculecomprising the gene into the T cells. In a preferred embodiment of theinvention, the T cells are contacted with the stimulatory agent at leastabout 2 hours before introducing the nucleic acid molecule into the Tcells. In another embodiment of the invention, the T cells are contactedwith the stimulatory agent at least about 4 hours before introducing thenucleic acid molecule into the T cells. In another embodiment of theinvention, the T cells are contacted with the stimulatory agent at leastabout 6 hours before introducing the nucleic acid molecule into the Tcells. In another embodiment of the invention, the T cells are contactedwith the stimulatory agent at least about 8 hours before introducing thenucleic acid molecule into the T cells. In other embodiments, the Tcells are contacted with the stimulatory agent at most about 2 hoursbefore transfection, at most about 4 hours before transfection, at mostabout 6 hours before transfection, at most about 8 hours beforetransfection, at most about 10 hours before transfection, at most about12 hours before transfection, at most about 14 hours beforetransfection, at most about 16 hours before transfection, at most about18 hours before transfection, at most about 20 hours beforetransfection, at most about 22 hours before transfection, at most about24 hours before transfection.

In an even more preferred embodiment of the invention, the T cells arecontacted with stimulating agent between about 1 hour and 24 hours priorto introducing the nucleic acid molecule into the T cells. In a mostpreferred embodiment of the invention, proliferating T cells arecontacted with at least one stimulatory agent between about 5 and 15hours, such as about 10 hours prior to transfecting a nucleic acidmolecule comprising a gene of interest.

In one embodiment of the method, proliferating T cells are contactedwith at least one stimulatory agent and further transfected with thenucleic acid molecule. In another embodiment of the method, thenon-proliferating T cells are stimulated to proliferate, then contactedwith at least one stimulatory agent prior to being transfected accordingto the method of the invention. Non-proliferating T cells can bestimulated to proliferate using agents well known in the art, such asthose described above, under the section “Stimulatory Agents”. Preferredagents include a combination of an agent which provides a primaryactivation signal and an agent which provides a costimulatory signal.Other preferred combinations of agents for stimulating proliferation ofT cells include combinations of a phorbol ester and a calcium ionophore,or PMA and IL-2.

According to a most preferred embodiment of the method for transfectingprimary T cells, resting primary T cells are first contacted with atleast one agent which stimulates proliferation of T cells, such as acombination of a calcium ionophore and a phorbol ester. At a timebetween approximately 3 and 8 hours, preferably approximately 5 hours,following induction of T cell proliferation, the proliferating T cellsare contacted with at least one agent which stimulates the T cells.Finally, between 2 and 15 hours, preferably about 10 hours, followingcontact with the stimulatory agent, the T cells are transfected with anucleic acid molecule comprising a gene of interest, such that the geneis expressed in the T cells. In another embodiment, the T cells arefurther contacted with agents that stimulate the T cells aftertransfection of the T cells.

To obtain primary T cells from a subject, peripheral blood mononuclearcells can be isolated from a subject and purified by density gradientcentrifugation, e.g., Ficoll/Hypaque. In a specific embodiment, thepurified peripheral blood cells are then transfected with a nucleic acidmolecule according to the method of the invention. In other embodimentsof the method, the peripheral blood mononuclear cells are furtherenriched in specific cell types prior to being transfected. Monocytescan be depleted, for example, by adherence on plastic.

If desired, the CD4⁺ T cell population can further be enriched byseparation from residual monocytes, B cells, NK cells and CD8⁺ T cellsusing monoclonal antibody (mAb) and anti-mouse-Ig coated magnetic beadsusing commercially available mAbs (such as anti-CD 14 (Mo2), anti-CD11b(Mo1), anti-CD20 (B1), anti-CD16 (3G8) and anti-CD8 (7PT 3F9) mAbs). Themethod of the invention can also be applied to subsets of CD4⁺ T cells,such as CD4⁺CD45RA⁺ (naive CD4⁺ T cells) and CD4^(+CD)45RO⁺ (memory Tcells) T cell subsets. These can be prepared as described above, withthe additional use of anti-CD45RO antibody (UCHLI) for the preparationof the CD4⁺CD45RA⁺cells and the addition of anti-CD45RA antibody (2H4)for the preparation of the CD4⁺CD45RO⁺ T cells.

The efficiency of the purification can be analyzed by flow cytometry(Coulter, EPICS Elite), using anti-CD3, anti-CD4, anti-CD8, anti-CD14mAbs, or additional antibodies that recognize specific subsets of Tcells, followed by fluorescein isothiocyanate conjugated goat anti mouseimmunoglobulin (Fisher, Pittsburgh, Pa.) or other secondary antibody.

In a preferred embodiment of the invention, the method of the inventionfurther comprises stimulating the T cells to expand in vitro aftertransfection of the T cells. T cells can be stimulated to expand invitro as described in the Examples section. In a specific embodiment, Tcells are incubated with an agent which provides a primary activatingsignal, such as anti-CD3 and an agent which provides a costimulatorysignal, such as an anti-CD28 antibody. Two days later, the cells arediluted with fresh medium and the agent providing the costimulatoryagent is added to the culture. The T cells are then counted, sized, anddiluted with fresh medium every two days until the sizing distributionshifted nearly back to a resting cell profile (at about 10 days). The Tcells can then be restimulated with the agent which provides a primaryactivating signal and an agent which provides a costimulatory signal. Tcells sizing and couting can be performed using a Coulter Counter, asdescribed herein.

In an even more preferred embodiment, the T cells are primary T cells.Thus, T cells can be obtained from a subject, transfected according tothe method of the invention, and expanded in vitro. In anotherembodiment of the invention, the transfected and expanded T cells arereadministered to the subject. It may be preferable to further purifythe T cells prior to administering into the subject, such as by gradientcentrifugation.

4. Transfection of the T Cells

The invention pertains to methods for transfecting T cells with anucleic acid comprising a gene, such that the gene is expressed in the Tcells. The language “transfecting T cells” is intended to include anymeans by which a nucleic acid molecule can be introduced into a T cell.The term “transfection” encompasses a variety of techniques useful forintroduction of nucleic acids into mammalian cells includingelectroporation, calciumphosphate precipitation, DEAE-dextran treatment,lipofection, microinjection, and viral infection. Suitable methods fortransfecting mammalian cells can be found in Sambrook et al. (MolecularCloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratorypress (1989)) and other laboratory textbooks.

In a preferred embodiment of the invention, the nucleic acid moleculeencoding a gene of interest is introduced into a T cell using a viralvector. Such viral vectors include, for example, recombinantretroviruses, adenovirus, adeno-associated virus, and herpes simplexvirus-1. Retrovirus vectors and adeno-associated virus vectors aregenerally understood to be the recombinant gene delivery system ofchoice for the transfer of exogenous genes in vivo, particularly intohumans. Alternatively they can be used for introducing exogenous genesex vivo into T cells. These vectors provide efficient delivery of genesinto T cells, and the transferred nucleic acids are stably integratedinto the chromosomal DNA of the host cell. A major prerequisite for theuse of retroviruses is to ensure the safety of their use, particularlywith regard to the possibility of the spread of wild-type virus in thecell population. The development of specialized cell lines (termed“packaging cells”) which produce only replication-defective retroviruseshas increased the utility of retroviruses for gene therapy, anddefective retroviruses are well characterized for use in gene transferfor gene therapy purposes (for a review see Miller, A. D. (1990) Blood76:271). Thus, recombinant retrovirus can be constructed in which partof the retroviral coding sequence (gag, pol, env) is replaced by a geneof interest rendering the retrovirus replication defective. Thereplication defective retrovirus is then packaged into virions which canbe used to infect a target cell through the use of a helper virus bystandard techniques. Protocols for producing recombinant retrovirusesand for infecting cells in vitro or in vivo with such viruses can befound in Current Protocols in Molecular Biology, Ausubel, F. M. et al.(eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 andother standard laboratory manuals. Examples of suitable retrovirusesinclude pLJ, pZIP, pWE and pEM which are well known to those skilled inthe art. Examples of suitable packaging virus lines for preparing bothecotropic and amphotropic retroviral systems include ψCrip, ψCre, ψ2 andψAm.

Furthermore, it has been shown that it is possible to limit theinfection spectrum of retroviruses and consequently of retroviral-basedvectors, by modifying the viral packaging proteins on the surface of theviral particle (see, for example PCT publications WO93/25234 andWO94/06920). For instance, strategies for the modification of theinfection spectrum of retroviral vectors include: coupling antibodiesspecific for cell surface antigens to the viral env protein (Roux et al.(1989) PNAS 86:9079-9083; Julan et al. (1992) J. Gen Virol 73:3251-3255;and Goud et al. (1983) Virology 163:251-254); or coupling cell surfacereceptor ligands to the viral env proteins (Neda et al. (1991) J BiolChem 266:14143-14146). Coupling can be in the form of the chemicalcross-linking with a protein or other variety (e.g. lactose to convertthe env protein to an asialoglycoprotein), as well as by generatingfusion proteins (e.g. single-chain antibody/env fusion proteins). Thus,in a specific embodiment of the invention, viral particles containing anucleic acid molecule containing a gene of interest operably linked toappropriate regulatory elements, are modified for example according tothe methods described above, such that they can specifically targetsubsets of T cells. For example, the viral particle can be coated withantibodies to surface molecule that are specific to certain types of Tcells. In particular, it is possible to selectively target CD4⁺ T cellsby linking to the viral particle antibodies that recognize the CD4molecule on the T cell. Thus, infection of CD4⁺ T cells will occurpreferentially over infection of CD8⁺ T cells. This method isparticularly useful when only specific subsets of T cells are desired tobe transfected. Additional retroviral systems for introducing andexpressing a nucleic acid molecule comprising a gene of interest in Tcells, including primary T cells, are described in Kasid, A. et al.(1990) Proc. Natl. Acad. Sci. U.S.A. 87, 473; Morecki, S. et al. (1991)Cancer Immunol. Immunother. 32, 342; Culver, K. et al. (1991) Proc.Natl. Acad. Sci. U.S.A. 88, 3155; and Finer, M. H. et al. (1994) Blood,83, 43.

Another viral gene delivery system useful in the present inventionutilitizes adenovirus-derived vectors. The genome of an adenovirus canbe manipulated such that it encodes and expresses a gene product ofinterest but is inactivated in terms of its ability to replicate in anormal lytic viral life cycle. See for example Berkner et al. (1988)BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434; andRosenfeld et al. (1992) Cell 68:143-155. Suitable adenoviral vectorsderived from the adenovirus strain Ad type 5 dl324 or other strains ofadenovirus (e.g., Ad2, Ad3, Ad7 etc.) are well known to those skilled inthe art. Recombinant adenoviruses can be advantageous in certaincircumstances in that they are not capable of infecting nondividingcells. Furthermore, the virus particle is relatively stable and amenableto purification and concentration, and as above, can be modified so asto affect the spectrum of infectivity. Additionally, introducedadenoviral DNA (and foreign DNA contained therein) is not integratedinto the genome of a host cell but remains episomal, thereby avoidingpotential problems that can occur as a result of insertional mutagenesisin situations where introduced DNA becomes integrated into the hostgenome (e.g., retroviral DNA). Moreover, the carrying capacity of theadenoviral genome for foreign DNA is large (up to 8 kilobases) relativeto other gene delivery vectors (Berkner et al. cited supra; Haj-Ahmandand Graham (1986) J. Virol. 57:267). Most replication-defectiveadenoviral vectors currently in use and therefore favored by the presentinvention are deleted for all or parts of the viral E1 and E3 genes butretain as much as 80% of the adenoviral genetic material (see, e.g.,Jones et al. (1979) Cell 16:683; Berkner et al., supra; and Graham etal. in Methods in Molecular Biology, E. J. Murray, Ed. (Humana, Clifton,N.J., 1991) vol. 7. pp. 109-127). Expression of the gene of interestcomprised in the nucleic acid molecule can be under control of, forexample, the E1A promoter, the major late promoter (MLP) and associatedleader sequences, the E3 promoter, or exogenously added promotersequences.

Yet another viral vector system useful for delivery of a nucleic acidmolecule comprising a gene of interest is the adeno-associated virus(AAV). Adeno-associated virus is a naturally occurring defective virusthat requires another virus, such as an adenovirus or a herpes virus, asa helper virus for efficient replication and a productive life cycle.(For a review see Muzyczka et al. Curr. Topics in Micro. and Immunol.(1992) 158:97-129). Adeno-associated virusses exhibit a high frequencyof stable integration (see for example Flotte et al. (1992) Am. J.Respir. Cell. Mol. Biol. 7:349-356; Samulski et al. (1989) J. Virol.63:3822-3828; and McLaughlin et al. (1989) J. Virol. 62:1963-1973).Vectors containing as few as 300 base pairs of AAV can be packaged andcan integrate. Space for exogenous DNA is limited to about 4.5 kb. AnAAV vector such as that described in Tratschin et al. (1985) Mol. Cell.Biol. 5:3251-3260 can be used to introduce DNA into T cells. A varietyof nucleic acids have been introduced into different cell types usingAAV vectors (see for example Hermonat et al. (1984) Proc. Natl. Acad.Sci. USA 81:6466-6470; Tratschin et al. (1985) Mol. Cell. Biol.4:2072-2081; Wondisford et al. (1988) Mol. Endocrinol. 2:32-39;Tratschin et al. (1984) J. Virol. 51:611-619; and Flotte et al. (1993)J. Biol. Chem. 268:3781-3790). Other viral vector systems that may haveapplication in gene therapy have been derived from herpes virus,vaccinia virus, and several RNA viruses.

In another embodiment of the invention, the nucleic acid moleculecomprising a gene of interest is introduced into a T cell bynon-viral-mediated methods of transfection well known in the art. Thesemethods include electroporation, calcium phosphate precipitation, andDEAE dextran transfection.

In yet another embodiment, the nucleic acid molecule comprising a geneof interest is carried by and delivered into a T cell by a cell-deliveryvehicle. Such vehicles include, for example, cationic liposomes(Lipofectin™) or derivatized (e.g. antibody conjugated) polylysineconjugates, gramicidin S, artificial viral envelopes. These vehicles candeliver a nucleic acid that is incorporated into a plasmid, vector, orviral DNA. In a specific embodiment, efficient introduction of thenucleic acid molecule in primary T lymphocytes is obtained bytransfecting the primary T lymphocytes with adeno-associated virusplasmid DNA complexed to cationic liposomes, as described in Philip, R.et al. (1994) Mol. Cell. Biol. 14, 2411.

In another embodiment of the invention, the nucleic acid moleculecomprising a gene of interest is delivered into a specific cell in theform of a soluble molecular complex. The complex contains the nucleicacid releasably bound to a carrier comprised of a nucleic acid bindingagent and a cell-specific binding agent which binds to a surfacemolecule of the specific T cell and is of a size that can besubsequently internalized by the cell. Such complexes are described inU.S. Pat. No. 5,166,320.

In another embodiment of the invention the nucleic acid is introducedinto T cells by particle bombardment, as described in Yang, N.-S. andSun, W. H. (1995) Nature Medicine 1, 481.

5. Nucleic Acid Molecules Comprising a Gene of Interest

The invention pertains to an improved method for introducing a nucleicacid molecule comprising a gene into a T cell. The language “a nucleicacid molecule” is intended to include DNA and RNA, and may be eithersingle or double-stranded. The term “gene” is intended to include a DNAnucleotide sequence that can be transcribed into RNA or alternatively,an RNA molecule that can be translated into at least one protein.

In a specific embodiment of the invention, the gene comprises anucleotide sequence containing one or more open reading frames, i.e.,sequences that code for peptides, such that upon transfection into the Tcell according to the method of the invention, at least one protein issynthesized in the T cell. The gene encoding at least one protein can beany gene, such as a gene encoding a cytokine. The gene can code for onepeptide or the gene can encode several peptides.

In another embodiment of the invention, the gene is a nucleotidesequence, which upon introduction in the T cells according to the methodof the invention is expressed into one or more functional RNA molecules(eg. an antisense RNA molecule). In a preferred embodiment of theinvention, the functional RNA molecule inhibits, or at least decreases,expression of one or more endogenous genes in the T cell. Thus, themethod of the invention is useful for decreasing expression of aselected gene in T cells. For example, T cells are transfected with anucleic acid molecule comprising a gene encoding antisense RNA, suchthat translation of an endogenous RNA is reduced. An “antisense” nucleicacid comprises a nucleotide sequence which is complementary to a “sense”nucleic acid, e.g., complementary to an mRNA sequence encoding aprotein, constructed according to the rules of Watson and Crick basepairing. Accordingly, an antisense nucleic acid can hydrogen bond to asense nucleic acid. The antisense sequence complementary to a sequenceof an mRNA can be complementary to a sequence found in the coding regionof the mRNA or can be complementary to a 5′ or 3′ untranslated region ofthe mRNA. Preferably, an antisense nucleic acid is complementary to aregion preceding or spanning the initiation codon or in the 3′untranslated region of an mRNA. For a discussion of the regulation ofgene expression using antisense genes see Weintraub, H. et al.,Antisense RNA as a molecular tool for genetic analysis, Reviews—Trendsin Genetics, Vol. 1(1) 1986.

In another embodiment of the invention, expression of an endogenous genein a T cell is reduced by introducing into the T cell a nucleic acidencoding a ribozyme according to the method of the invention. Ribozymesare catalytic RNA molecules with ribonuclease activity which are capableof cleaving a single-stranded nucleic acid, such as an mRNA, to whichthey have a complementary region. A ribozyme having specificity for anucleic acid of interest can be designed based upon the nucleotidesequence of the nucleic acid. For example, a derivative of a TetrahymenaL-19 IVS RNA can be constructed in which the base sequence of the activesite is complementary to the base sequence to be cleaved in an mRNA ofinterest. See for example Cech et al. U.S. Pat. No. 4,987,071; and Cechet al. U.S. Pat. No. 5,116,742.

The “nucleic acid molecule” comprising the gene can be a DNA molecule oran RNA molecule. The nucleic acid molecule can be a portion of a naturalnucleic acid molecule, or alternatively, it can be made synthetically.The nucleic acid molecule typically contains regulatory elements towhich the gene is operably linked. “Operably linked” is intended to meanthat the nucleotide sequence of the gene is linked to at least oneregulatory sequence in a manner which allows for expression (i.e.,transcription) of the gene in T cells. Regulatory sequences areart-recognized and are selected to direct expression of the gene in anappropriate T cell. Accordingly, the term regulatory sequence includespromoters, enhancers and other expression control elements. Suchregulatory sequences are known to those skilled in the art and arefurther described in Goeddel, Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif. (1990).

These regulatory elements include those required for transcription andtranslation of the gene, and may include promoters, enhancers,polyadenylation signals, and sequences necessary for transport of themolecule to the appropriate cellular compartment. When the nucleic acidis a cDNA in a recombinant expression vector, the regulatory functionsresponsible for transcription and/or translation of the cDNA are oftenprovided by viral sequences. Examples of commonly used viral promotersinclude those derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40, and retroviral LTRs.

Regulatory sequences linked to the cDNA can be selected to provideconstitutive or inducible transcription, by, for example, use of aninducible enhancer. Thus, in a specific embodiment of the invention thenucleic acid molecule-comprising a gene of interest is under the controlof an inducible control element, such that expression of the gene can beturned on or off, by contacting or not contacting, respectively, the Tcells containing the nucleic acid with an agent which affects theinducible control element.

In a specific embodiment, the nucleic acid molecule is under the controlof an inducible control element. Inducible regulatory systems for use inmammalian cells are known in the art, for example systems in which geneexpression is regulated by heavy metal ions (Mayo et al. (1982) Cell29:99-108; Brinster et al. (1982) Nature 296:39-42; Searle et al. (1985)Mol. Cell. Biol. 5:1480-1489), heat shock (Nouer et al. (1991) in HeatShock Response, e.d. Nouer, L., CRC, Boca Raton, Fla., pp167-220),hormones (Lee et al. (1981) Nature 294:228-232; Hynes et al. (1981)Proc. Natl. Acad. Sci. USA 78:2038-2042; Klock et al. (1987) Nature329:734-736; Israel & Kaufman (1989) Nuc. Acids Res. 11:2589-2604 andPCT Publication No. WO 93/23431), tetracycline (Gossen, M. and Bujard,H. (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551 and PCT PublicationNo. WO 94/29442) or FK506 related molecules (PCT Publication No.WO94/18317).

Inducible control elements can be inducible in all T cells, oralternatively only in a specific subset of T cells, such as in CD4⁺ Tcells, CD8⁺ T cells, T helper 1 (Th1), T helper 2 (Th2) cells. Induciblecontrol elements could also be elements which are inducible by one agentin one type of T cells, (such as CD4⁺ T cells) and which are inducibleby another agent in another type of T cells (such as CD8⁺ T cells).

In another embodiment of the invention, the nucleic acid moleculecomprising a gene of interest is under the control of regulatorysequences which constitutively drive the expression of the nucleic acidmolecule. Regulatory elements which drive constitutive expression ofnucleic acid molecules to which they are operably linked can be viralelements (e.g. derived from polyoma, Adenovirus 2, cytomegalovirus,Simian Virus 40 or retrovirus). Alternatively, constitutive T cellenhancers can be used such as a T cell receptor enhancer (see e.g.,Winoto and Baltimore (1989) EMBO J. 8:729-733).

The nucleic acid molecule comprising a gene of interest operably linkedto regulatory elements is typically carried within a vector (e.g. aplasmid or viral DNA) which includes sequences that are necessary for invitro selection and amplification of the vector in bacteria. A vectorallowing for the expression of the gene carried by the vector isreferred to herein as an “expression vector”.

6. Applications for the Method of the Invention

The invention pertains to improved methods for introducing andexpressing a gene comprised in a nucleic acid molecule into T cells. Ina preferred embodiment of the invention, the T cells are primary Tcells. Thus, the method of the invention allows for high levelexpression of a gene when introduced into primary T cells, as comparedto previous methods for transfecting primary T cells. The ability totransfect primary T cells with a nucleic acid molecule comprising agene, such that the gene is expressed in the T cells has numerousapplications, in particular for gene therapy.

In one specific embodiment, peripheral blood T cells are obtained from asubject and transfected ex vivo with a nucleic acid molecule containinga gene encoding a protein of interest, such that the protein issynthesized in the T cells. The T cells may further be readministered tothe subject. In a specific embodiment, the exogenous protein synthesizedin the T cell is secreted by the T cell. Thus, the invention provides amethod for producing in an individual a secretable protein. Proteinswithin the scope of the invention include, for example, cytokines,lymphokines, growth factors. Thus, the proteins produced by thetransfected T cell may be predominantly targeted to other cells than toT lymphocytes themselves.

Alternatively, the protein produced by the transfected T cell is anintracellular or membrane protein. In a specific embodiment, theexogenous protein is a protein that protects the T cells from aninfection by, for example, a virus. Such a method is useful forexpanding a population of T cells of which some are infected with avirus, such as human immunodeficiency virus (HIV). Thus, the populationof T cells can be expanded without concomittant spread of the infectionto all cells.

In another embodiment, the exogenous protein is a protein which kills aspecific subset of T cells, such as a toxin. The protein can beselectively targeted to specific subsets of T cells by having the geneunder the control of a regulatory control element specific for thatsubset of T cells. It is also possible to target an exogenous genespecifically to certain types of T cells by using a transfection methodthat allows for selective transfection of certain T cell subsets. Forexample, T cells can be transfected with liposomes containing on theirmembrane a ligand for a T cell subset specific molecule.

The gene introduced into the T cell by the method of the invention canalso be a gene designed to treat a genetic disease, such as severecombined immunodeficiency due to adenine deaminase deficiency. Forexample, a gene encoding adenosine deaminase can be introduced into, anexpressed in, primary T lymphocytes using the method of the invention.Another embodiment of the invention pertains to the treatment ofhyper-IgM syndrome, a genetic disorder characterized by a mutation inthe CD40 ligand (gp39) on T cells and characterized by defects in helperT cell-dependent antibody responses. Thus, T cells from a subject havinghyper-IgM syndrome can be transfected ex vivo with a nucleic acidencoding gp39, preferably under the control of its own promoter,followed by administration of the T cells to the patient. Other geneticdiseases resulting from a dysfunctional gene in T cells, such as a geneencoding a protein involved in T cell signal transduction pathways, canbe treated by the method of the invention.

The invention is further illustrated by the following examples, whichshould not be construed as further limiting. The contents of allreferences, pending patent applications and published patents, citedthroughout this application are hereby expressly incorporated byreference.

EXAMPLES Example 1 In vitro Long-term Culture of CD28⁺ Peripheral BloodT Lymphocytes

Direct transfection experiments are often required to demonstrate thefunctional importance of putative regulatory elements in vivo. Suchstudies typically utilize transformed or immortalized cell lines.However, it would be preferable to study regulatory DNA sequences in theprimary cells of interest. For the prospective study of cellcycle-regulated, especially G₀-specific, gene expression, themaintenance of a physiological background is required. These exampleswere designed to develop conditions allowing for expression of exogenousDNA transfected into primary T cells.

Peripheral blood T cells were prepared as follows. Buffy coats wereobtained by leukophoresis of healthy donors aged 21 to 31 years or fromthe Red Cross. Peripheral blood mononuclear cells (PBMCs) were obtainedby density gradient centrifugation through a Ficoll-Hypaque (Pharmacia)cushion or with Lymphocyte Separation Medium (Litton Bionetics). TheCD28⁺ subset of T cells was then isolated from the PBMCs by negativeselection using immunoadsorption with goat anti-mouse Ig-coated magneticparticles (Dynal Inc.) as previously described (June, C. H., Ledbetter,J. A., Gillespie, M. M., Lindsten, T., and Thompson, C. B. (1987) Mol.Cell. Biol. 11:5435-5445). Cell purification was routinely monitored byflow cytometry and histochemistry. The resulting cell populationwas >99% CD2⁺ and >98% CD28⁺ as measured by fluorescence-activated cellsorter (FACS) analysis using fluorescein isothiocyanate(FITC)-conjugated mAbs. Monocytes, B cells, and large granularlymphocytes were not detectable by immunofluorescence analysis.Alternatively, resting T cells were prepared from the mononuclear cellfraction by centrifugal elutriation as previously described (Thompson,C. B., Ryan, J. J., Sieckmann, D. G., Finkelman, F. D., Mond, J. J., andScher, I. (1983) J. Immunol. Methods 63:299-307). These cells were >95%CD2⁺ as determined by flow cytometry. With both methods, cell viabilitywas >99% as measured by trypan blue exclusion.

Human peripheral blood T cells obtained as described above were shown tobe >99% CD2⁺, >98% CD28⁺ and in a quiescent state. The purified T cellsused in this study were depleted of accessory cells and did notproliferate in vitro after stimulation with phytohemagglutinin (PHA),phorbol myristate acetate (PMA), or ionomycin alone. However, these Tcells could be stimulated to divide by cross-linking the TCR-CD3 complexwith immobilized monoclonal antibody (mAb) or by using appropriateamounts of PMA and ionomycin (Lindsten, T., June, C. H., and Thompson,C. B. (1988) EMBO J. 7:2787-2794). Under these conditions, >90% of theresting T cells were activated and the majority of the cellssynchronously proceeded through one round of cell division.

To activate resting T cells and promote their long-term expansion inculture, freshly isolated resting T cells, obtained as described above,were cultured at a concentration of 2×10⁶ cells/ml in complete medium:RPMI 1640 (GIBCO), supplemented with 10% heat-inactivated fetal calfserum (GIBCO), 2 mM L-glutamine, penicillin G (100 U/ml), streptomycin(100 mg/ml), and 15 mM Hepes(N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid; pH 7.4; GIBCO);and rested overnight at 37° C., 5% CO₂. Following overnight incubation,on Day 1of the expansion protocol, resting T cells were stimulated witha saturating quantity of immobilized anti-CD3 antibody (αCD3) mAb G19-4directed against the CD3ε chain in the presence of soluble anti-CD28antibody (αCD28) mAb 9.3 (1 μg/ml) as described by June et al., (1987)Mol. Cell. Biol. 11:5435-5445. CD3 mAb G19-4 (IgG1) was produced andpurified as described previously (Ledbetter, J. A., Martin, P. J.,Spooner, C. E., Wofsy, D., Tsu, T. T., Beatty, P. G., and Gladstone, P.(1985) J. Immunol. 135:2331-2336). mAb G19-4 was absorbed to the surfaceof plastic tissue culture flasks/plates as previously described(Geppert, T. D., and Lipsky, P. E. (1987) J. Immunol. 138:1660-1666) inamounts appropriate for proliferation. This was done because of therequirement for cross-linking (Williams, J. M., Ransil, B. J., Shapiro,H. M., and Strom, T. B. (1984) J. Immunol. 133:2986-2995) and to preventinternalization of the CD3 complex (Ledbetter, J. A., June, C. H.,Martin, P. J., Spooner, C. E., Hansen, J. A., and Meier, K. E. (1986) J.Immunol. 136:3945-3952). CD28 mAb 9.3 (IgG2a) was purified on proteinA-sepharose, dialyzed against PBS, filtered through a 0.22 μm sterilefilter, cleared of aggregates by centrifugation (100,000×g for 45 min)and used at 1 μg/ml (Ledbetter, J. A., Martin, P. J., Spooner, C. E.,Wofsy, D., Tsu, T. T., Beatty, P. G., and Gladstone, P. (1985) J.Immunol. 135:2331-2336).

Two days later, on Day 3, the activated T cells were counted, sized, anddiluted to a concentration of 0.5×10⁶ cells/ml with fresh completemedium. mAb 9.3 was added to a final concentration of 0.5 μg/ml.Counting, sizing, and dilution of cells were repeated every 2 days untilthe sizing distribution shifted nearly back to a resting cell profile atwhich point T cells were resuspended in complete medium at 2×10⁶cells/ml and restimulated as above (first restimulation usually aroundDay 10).

Cells were counted using a Coulter ZM Counter (Coulter Electronics).Cells were sized on a linear scale with a Coulter Counter model ZMequipped with a cylindrical 70-μm aperture and a Channelyzer model C-256(Coulter Electronics) interfaced to an IBM PC computer. Cells weresuspended in Isoton and calibration was performed using latex beads ofuniform diameters.

Treatment of mitogen or anti-T cell receptor (anti-TCR) stimulated Tcells with αCD28 induced a synergistic increase in T cell proliferation.Costimulation of resting T cells with αCD3 plus αCD28 resulted in aninitial period of vigorous exponential growth and cellular metabolismcharacterized by cellular enlargement, clumping, and acidification ofthe culture medium. Cells proceeded through several rounds of celldivision and increased in number between 6-8 fold over the course of thefirst 7 to 8 days in culture. At this point, their growth ratedecreased. By day 10-11 of culture, cell division ceased and cellsresembled resting cells based on their size (FIG. 1). At this point inthe expansion protocol, cells were restimulated with immobilized αCD3 inthe presence of αCD28 and experienced another period of exponentialgrowth characterized by cellular aggregation and enlargement.

FIG. 2 illustrates the growth characteristics of cells cultured in thismanner. As demonstrated, cells could be maintained and grown inexponential fashion for many weeks (more than 3 months) using repeatedαCD3/αCD28 costimulation. Flow cytometric analysis was performed atvarious time points to follow the phenotypic evolution of these cells.With time, T cells expanded in this fashion became progressively moreCD4⁺45RO⁺ reflecting a switch in phenotype from naive T helper (Th)cells to memory cells. This is in direct contrast to cells which weregrown in the presence of exogenous IL-2 after αCD3/αCD28 costimulation.These cells became progressively more CD8⁺ with time and were eventuallyincapable of further proliferation in culture. These observations showthat some factor produced by CD4⁺ cells is required for continuous Tcell proliferation.

Example 2 Cellular Proliferation is not Sufficient for Expression ofExogenous DNA

Having established repeated αCD3/αCD28 costimulation for the long-termclonal expansion of primary T cells, cells grown using this protocolwere analyzed for endogenous ets-1 mRNA expression by Northern blotanalysis.

For RNA extraction, cells were harvested by centrifugation and totalcellular RNA extracted using guanidinium isothiocyanate (Chirgwin etal., 1979). The samples were equalized for rRNA, and the equalizationconfirmed by ethidium bromide staining of equal amounts of the RNAsamples separated on a nondenaturing 1% agarose gel as describedpreviously (June, C. H., Ledbetter, J. A., Gillespie, M. M., Lindsten,T., and Thompson, C. B. (1987) Mol. Cell. Biol. 11:5435-5445). Theseequalized RNA samples (5 to 10 μg) were separated on 1%agarose/formaldehyde gels and transferred to nitrocellulose. DNA probeswere labeled by nick translation to a specific activity of 3 to 9×10⁸cpm/μg. The IL-2 specific probe was a 1.0 kb PstI cDNA insert derivedfrom the pTCGF5 plasmid (Clark, S. C., Arya, S. K., Wong-Staal, F.,Matsumoto-Kobayashi, M., Kay, R. M., Kaufman, R. J., Brown, E. L.,Shoemaker, C., Copeland, T., and Oroszian, S. et al. (1984) Proc. Natl.Acad. Sci. USA 81:2543-2547). The HLA B7 probe was a 1.4-kb PstIfragment isolated from pHLA-B7 (Sood, A. K., Pereira, D., and Weissman,S. M. (1981) Proc. Natl. Acad. Sci. USA 78:616-620). The ets-1 DNA probeconsisted of a 442 base pair EcoRI/XbaI fragment from the 5′ end of a1.9-kb ets-1 cDNA (Ho, I-C., Bhat, N. K., Gottschalk, L. R., Lindsten,T., Thompson, C. B., Papas, T. S., and Leiden, J. M. (1990) Science250:814-817). The membranes were washed and exposed to x-ray film (KodakXAR-2 or XAR-5) for 4 hours to 7 days at −70° C. using intensifyingscreens.

For preparing Northern blots, RNA was extracted from peripheral blood Tcells cultured according to the protocol allowing for long term clonalexpansion of primary T cells described above at various time pointsafter activation with αCD3/αCD28 on Day 1or after restimulation on Day8. The results of the Northern blot analysis are presented in FIG. 3.Resting human T cells express high levels of ets-1 mRNA and protein.Resting T cells and “activated” cells on Day 8 expressed high levels ofets-1 mRNA. Upon antigen-receptor cross-linking in the presence ofαCD28, ets-1 mRNA decreased to undetectable levels by 6 hours. In bothcells from Day 1 and Day 8, ets-1 mRNA was reexpressed and maintainedbetween 24 and 72 hours following stimulation.

To determine which cis-acting regulatory elements modulate ets-1 geneexpression, primary cells in log phase growth were transfected on Day 6of the long-term culture protocol with a plasmid containing the ets-1promoter linked to the CAT gene (ETS-1-CAT-2). This construct exhibitedrobust reporter activity in Jurkat T cells. Following transfection ofthe primary T cell with 1 μg DNA with either DEAE-dextran (Ho, I-C.,Bhat, N. K., Gottschalk, L. R., Lindsten, T., Thompson, C. B., Papas, T.S., and Leiden, J. M. (1990) Science 250:814-817), the cells wererepeatedly washed then resuspended in complete medium. 40 hours aftertransfection, cells were harvested and assayed for CAT activity.

With the DEAE-dextran transfection protocol, cells were washed once withPBS and then once with TS buffer pH 7.4. After the second wash, cellswere resuspended at 10⁷/ml in TS buffer containing 500 μg ofDEAE-dextran (molecular weight 500,000) and 1 to 10 μg of supercoiledplasmid DNA. This mixture was allowed to sit for 12 to 15 min at roomtemperature with occasional swirling. 10 mls of RPMI 1640 supplementedwith 20% heat-inactivated fetal calf serum, 2 mM L-glutamine, and 15 mMHepes (RPMI 1640/20% FCS/G/H) was added to the cells. The cells weretransferred to tissue culture flasks and incubated for 30 min at 37° C.,5% CO₂. The cells were then pelleted, washed once with RPMI 1640/20%FCS/G/H, and resuspended in 10 ml RPMI 1640/20% FCS/G/H at 37° C., 5%CO₂.

In other examples described herein, primary T cells are transfected byelectroporation. With the electroporation protocol, cells were washedtwice with ice-cold PBS and resuspended at 20×10⁶ cells/ml in ice-coldRPMI 1640/20% FCS/G/H. 6×10⁶ cells in 300 μl were transferred to asterile 0.4 cm electroporation cuvette (BioRad). 1 to 10 μg of reporterplasmid was added and the cells electroporated using a gene pulser(BioRad) at 250 V and 960 μfarads. The cells were incubated 10 min onice, diluted to 10 ml with RPMI 1640/20% FCS/G/H and placed at 37° C.,5% CO₂.

Equal volumes of cell extracts were assayed for CAT activity in a 16hour reaction. EDTA was added to a final concentration of 5 mM and theextracts heated at 65° C. for 10 min to prevent the hydrolysis ofacetyl-CoA and the deacetylation of chloramphenicol (Crabb, D. W., andDixon, J. E. (1987) Anal. Biochem. 163:88-92). Before autoradiography ofthe CAT assay thin-layer chromatography (TLC) plate, spots correspondingto [¹⁴C]chloramphenicol and its acetylated derivatives were quantitatedusing a Betascope (Betagen) or phosphorimager (Molecular Dynamics). Thepercent acetylation was calculated after subtracting background valuesfrom experimental acetylated and non-acetylated values. If the percentacetylation was out of the linear range of the assay for a given set oftransfections, equal volumes of cell extracts were diluted, and the CATassay reperformed. CAT activity was then normalized to the amount ofprotein in the reaction. Normalized CAT activity is expressed as(percent acetylation/mg protein)×50. All comparisons of reporteractivity derive from cells stimulated, transfected, harvested, andassayed at the same time with the same reagents.

No ETS-1-CAT-2 reporter activity was detected in primary cellstransfected in this manner. Since endogenous ets-1 mRNA ispreferentially expressed in resting cells and is reinduced approximately2-3 days following T-cell stimulation, ETS-1-CAT-2 reporter expressionwas expected, whether or not T cells reentered a resting state.Surprisingly, transfection of cells with positive controls such asRSV-CAT, HIV-1-CAT, and HTLV-1-CAT, also yielded no demonstrablereporter activity. Based on increases in cell number, cells at Day 6 ofthe long-term culture protocol were in log-phase growth (FIG. 2).However, transfection of these cells with constitutive reporterconstructs resulted in no detectable reporter activity suggesting thatproliferation alone is insufficient for efficient transgene expression.

To determine whether “active” signal transduction is required forreporter gene expression, primary T cells in log phase growth on Day 5of the culture protocol were stimulated with phorbol ester (PDBU) pluscalcium ionophore (ionomycin) 10 hours before transfection.

FIG. 4 depicts the timetable used for this and subsequent transfections.Resting T cells were stimulated to proliferate by incubation with asaturating amount of immobilized anti-CD3 antibody and anti-CD28 at 1μg/ml. Two days later, on Day 3, the activated T cells were counted,sized, and diluted to a concentration of 0.5×10⁶ cells/ml with freshcomplete medium. mAb 9.3 was added to a final concentration of 0.5μg/ml. At day 5, cells were stimulated with phorbol-12,13-dibutyrate(PDBU; from LC Services Corp.) at 10 ng/ml and ionomycin (Calbiochem) at0.4 μg/ml. On Day 6, 10 hours after stimulation, cells were transfectedwith 10 μg of constitutively expressed reporter construct RSV-CAT usingDEAE-dextran essentially as previously described (Ho, I-C., Bhat, N. K.,Gottschalk, L. R., Lindsten, T., Thompson, C. B., Papas, T. S., andLeiden, J. M. (1990) Science 250:814-817). pRSV-CAT (RSV-CAT) consistsof RSV LTR sequences fused to the 5′ end of coding sequences for CAT(Gorman, C. M., Merlino, G. T., Willingham, M. C., Pastan, I., andHoward, B. H. (1982) Proc. Natl. Acad. Sci. USA 79:6777-6781). Cellswere harvested 40 hours later and assayed for CAT activity.

The results are presented in FIG. 5. PDBU+ionomycin prestimulation ofproliferating primary cells resulted in a 67-fold increase in RSV-CATreporter expression relative to cells treated with conditioned mediumalone. To determine whether this dramatic difference in RSV-CAT reporteractivity was due to a difference in the proliferative capacity ofstimulated versus non-stimulated cells, the proliferative status ofthese cells was measured using the following: 1) acridine orangestaining for cell cycle analysis; 2) tritiated thymidine [³H]TdRincorporation as a measure of DNA synthesis; and 3) cell sizing as ageneral measure of cellular activation. Autologous resting primary cellsand αCD3/αCD28 stimulated cells from Day 3 of the long-term cultureprotocol were measured simultaneously as controls for the quiescentstate (G₀/G₁ interface) and robust proliferation.

Purified resting T cells (Day 1) were stimulated with a saturatingquantity of immobilized αCD3 mAb G19-4 in the presence of the αCD28 mAb9.3 (1 μg/ml). On Day 3, activated T cells were diluted to aconcentration of 0.5×10⁶/ml with fresh complete medium and mAb 9.3 addedto a final concentration of 0.5 μg/ml. On Day 6, T cells in exponentialgrowth were treated with phorbol-12,13-dibutyrate (PDBU) (10 ng/ml) plusionomycin (0.4 μg/ml) or conditioned medium alone for 10 hr. Cells fromDay 3 and Day 6 were stained with acridine orange for cell cycleanalysis as described below. Unstimulated cells (Day 1) were analyzedsimultaneously to determine the G₀/G₁ interface.

Cells were analyzed for DNA and RNA content on a FACScan flow cytometer(Becton-Dickinson) after staining with acridine orange (Polysciences)using a procedure described by Darzynkiewicz (1990) Methods Cell Biol.33:285-298). 1 to 5×10⁶ cells were washed two times with PBS and fixedin cold 70% ethanol at a concentration of 2×10⁶/ml. Cells werecentrifuged, washed, and resuspended in complete medium at aconcentration below 2×10⁶/ml. 0.2 ml of this cell suspension was stainedwith acridine orange and analyzed on the FACScan. Cells with increasedRNA content and unchanged DNA content were considered G₁ phase cells.Cells with increased RNA and DNA content were considered in S or G₂Mphases.

For determining [³H]TdR incorporation of T cells from Days 1, 3, and 6,cells were cultured in quadruplicate samples in flat-bottom 96-wellmicrotiter plates (Costar) at 5×10⁵ cells/well. The final culture volumewas 200 μl in complete medium. 1 μCi of tritiated thymidine [³H]TdR(ICN) was added to each well and the cultures incubated for 6 hours at37° C., 5% CO₂. After 6 hours of culture the cells were harvested ontoglass microfiber strips (Whatman) using a PHD cell harvester (CambridgeTechnologies) and counted in a liquid scintillation counter (LKB). Allvalues are expressed as the mean cpm±standard deviation of quadruplicatecultures.

As shown in FIG. 6, stimulation of resting T cells with αCD3/αCD28resulted in progression of greater than 92% of the cells from G₀ to G₁or S/G₂M phases of the cell cycle by the third day of cellularexpansion. This corresponded to a 207-fold increase in tritiatedthymidine incorporation and an increase in mean cellular volume. By Day6 of culture, greater than 91% of cells growing in conditioned mediumalone were in either G₁ or S/G₂M phases of the cell cycle. Greater than92% of PDBU+ionomycin treated cells assayed for RNA and DNA content 10hours after stimulation were found to be in G₁ or S/G₂M phases of thecell cycle. These data illustrate the actively cycling nature of cellsat the time of transfection and the equivalent proliferative capacitiesof PDBU/IONO stimulated versus non-stimulated cells. Indeed, PDBU/IONOstimulated cells did not demonstrate any increase in the rate of DNAsynthesis (35×10³ cpm versus 52×10³ cpm [³H]TdR incorporation) or meancellular volume when compared to their non-stimulated counterparts.Thus, no differences in the proliferative capacities of these two cellpopulations were found at the time of transfection which would accountfor differences in RSV-CAT reporter gene expression.

The Rous sarcoma virus LTR contains a calcium/calmodulin-dependentprotein kinase (CaM-kinase) response element which is capable ofconferring selective induction of transcription by activated CaM-kinasein the presence of elevated levels of calcium ions (Kapiloff, M. S.,Mathis, J. M., Nelson, C. A., Lin, C. R., and Rosenfeld, M. G. (1991)Proc. Natl. Acad. Sci. USA 88:3710-3714). To determine whetherdifferences in RSV-CAT expression between PDBU/IONO stimulated andnon-stimulated cells arose from specific trans-activation of the RSV LTRfollowing stimulation, cells were either prestimulated with PDBU/IONO ortreated with conditioned medium alone, transfected, then eitherimmediately cultured in conditioned medium or complete medium. 30 hoursafter transfection, cells cultured in complete medium were eitherstimulated with PDBU/IONO, αCD3/αCD28, or were treated with mediumalone. 10 hours later, cells were harvested for CAT activity. If theonly role of signal transduction is to activate transcription of the RSVLTR, then cells stimulated after transfection should also demonstrateincreased reporter activity. As shown in FIG. 7, PDBU/IONOprestimulation of cells resulted in a 345-fold increase in RSV-CATreporter activity relative to the non-stimulated proliferating control.Stimulation of cells 30 hours after transfection with either αCD3/αCD28or PDBU/IONO resulted in a small 3- or 3.5-fold increase in RSV-CATreporter activity, respectively. Culturing of transfected cells ingrowth-competent conditioned medium resulted in a 2.5-fold increase inCAT activity. In addition, PDBU/IONO or αCD3/αCD28 stimulation of cellsimmediately after transfection resulted in a small 4- to 5-fold increasein RSV-CAT activity. These data demonstrate that stimulation beforetransfection is required for RSV-CAT activity and suggest signaltransduction at the time of transfection facilitates reporter geneexpression either by increasing transfection efficiency or by renderingthe transgene competent for expression. Using this reporter construct,stimulation of cells post-transfection did not result in an appreciableincrease in reporter gene expression.

In the following example, it was shown that stimulation of primary Tcells with anti-CD3 and anti-CD28 10 hours prior to transfection alsoresulted in greatly enhanced expression of the reporter construct.HIV-1-CAT which has been described previously (Nabel, G., and Baltimore,D. (1987) Nature 326:711-713), was used in this example. Proliferating Tcells were prestimulated with either PDBU/IONO, αCD3/αCD28, or treatedwith conditioned medium alone, transfected with HIV-1-CAT, then eitherimmediately cultured in conditioned medium or stimulated 30 hours aftertransfection with PDBU/IONO, αCD3/αCD28, or medium alone. 10 hourslater, the cells were harvested and assayed for CAT activity. Results ofthe CAT assays are shown in FIG. 8. The results indicate thatprestimulation of primary T cells 10 hours prior to transfection greatlyenhances expression of the HIV-1-CAT reporter construct compared totransfection without prestimulation. Thus, the enhancement of expressionof the reporter construct is not dependent on the type of promoter andenhancer in the construct. Moreover, these results indicate thatprestimulation of the primary T cells to enhance expression of thetransfected reporter construct can also be done with a combination ofanti-CD3 and anti-CD28 antibodies.

Example 3 Expression of Exogenous DNA Requires TCR-dependent SignalTransduction at the Time of Transfection

To further dissect the requirements for efficient transgene expressionin primary T cells, the extensively studied and well characterizedpromoter/enhancer of the cellular IL-2 gene was used in transfectionexperiments. Northern blot analysis was used to characterize thekinetics of IL-2 gene expression after stimulation of the TCR-CD3complex by optimal amounts of immobilized αCD3. In addition, thesupernatants from these cultures were also analyzed for IL-2 content andthe cells analyzed for cell cycle progression.

The results are presented in FIG. 9. Panel B indicates that in thepresence of optimal αCD3 stimulation, IL-2 mRNA expression peaked at 6hours of culture; by 12 hours of culture IL-2 mRNA levels had decreasedto undetectable levels. This transient induction of IL-2 mRNA expressionwas accompanied by a small amount of IL-2 in the culture supernatant (5U/ml at 24 hours) and vigorous proliferation (41% of cells in S/G₂Mphases of the cell cycle by 48 hours) (Panel D). To summarize,stimulation of the TCR-CD3 complex resulted in the transient inductionof IL-2 gene transcription peaking 6 hours and decreasing toundetectable levels by 12 hours post-stimulation.

Given the inducible and transient nature of IL-2 mRNA transcription, therequirement of signal transduction at the time of transfection forIL2-CAT reporter gene expression was tested. The pIL2 CAT plasmid(IL2-CAT) contains the IL-2 promoter/enhancer (−585 to +18) immediately5′ of the chloramphenicol acetyltransferase (CAT) gene and has beendescribed previously (Bielinska, A., Shivdasani, R. A., Zhang, L., andNabel, G. J. (1990) Science 250:997-1000). Proliferating primary T cellson Day 5 of the expansion protocol were either prestimulated withPDBU/IONO or treated with conditioned medium alone, transfected with theIL2-CAT reporter plasmid, then immediately cultured in growth competentconditioned medium or complete medium. 30 hours after transfection,cells cultured in complete medium were stimulated with either PDBU/IONO,αCD3/αCD28, or were treated with medium alone. 10 hours later, cellnumber and viability were determined by trypan blue exclusion, and cellsharvested for CAT activity.

The CAT assay results are shown in FIG. 10 and are summarized asfollows: 1) In the absence of prestimulation, proliferating primarycells expressed extremely low levels of IL2-CAT reporter; 2) Stimulationof cells 30 hours after transfection did not increase this low level ofIL2-CAT reporter gene expression; 3) Following PDBU/IONO prestimulationand transfection, cells resuspended in lymphokine-rich conditionedmedium or complete medium also expressed extremely low levels of IL2-CATreporter; 4) However, following both PDBU/IONO prestimulation andTCR-directed stimulation 30 hours after transfection, IL2-CAT reporteractivity increased 79-fold relative to cells which only receivedprestimulation and approximately 20-fold relative to the non-stimulatedproliferating control. Therefore, IL2-CAT reporter gene expressionrequired TCR-dependent signal transduction both before and aftertransfection. These data are consistent with a model in which theintroduction of the IL2-CAT DNA reporter construct into an expressiblecompartment or state requires TCR-dependent signal transduction beforetransfection. However, even though the IL2-CAT reporter plasmid iscompetent for expression, little or no IL2-CAT transcription occurssince the initial signal transduction was delivered 10 hours beforetransfection and by the above Northern analysis (FIG. 9), IL-2 mRNAtranscription decreases to low levels by this timepoint.TCR/CD3-dependent stimulation after transfection results intrans-activation of the expression-competent IL-2 promoter/enhancer andCAT mRNA transcription with resulting reporter activity.

Example 4 Cellular Proliferation is Required for Transgene Expression

To address the role of cellular proliferation in transgene expression,freshly isolated resting T cells were stimulated with either αCD28 (1μg/ml), SEA (10 ng/ml, Toxin Technologies), PDBU (10 ng/ml)/IONO (0.4μg/ml), or medium alone for 10 hours. These cells were transfected withRSV-CAT, then harvested 40 hours later for CAT activity. Activated Tcells undergo cell division between 24-36 hours after mitogenicstimulation. Therefore, at the time of transfection, very few, if any, Tcells have progressed through M phase. By 40 hours post-transfection(approximately 50 hours post-stimulation), T cells stimulated with SEAor PDBU/IONO did exhibit phenotypic changes associated withproliferation (cellular enlargement, aggregation). However, thesepopulations had not increased in cell number. As shown in FIG. 11, thenormalized CAT activities for all these transfection conditions wererelatively low (0.07-0.26). Resting cells stimulated with PDBU/IONOdemonstrated a small 3.7-fold increase in RSV-CAT activity relative tothe resting control. This small increase in CAT activity betweenPDBU/IONO stimulated and resting cells may simply reflect the effects ofcellular activation and proliferation on RSV LTR transcription once thisreporter plasmid is capable of expression as a result of the initialsignal transduction. Of note, the CAT activities of cells stimulatedwith αCD28 or SEA were no different from that of resting cells. NeitherαCD28 nor SEA alone constitute complete mitogenic stimuli. The inductionof T-cell proliferation by SEA requires the addition of a secondcostimulatory signal provided by accessory cells (a requirement formajor histocompatibility complex (MHC) Class II presentation) or ofmonoclonal antibody stimulation of CD28 (Green, J. M., Turka, L. A.,June, C. H., and Thompson, C. B. (1992) Cell. Immunol. 145(1):11-20).Resting T cells do not express MHC Class II (HLA-Dr) on their surface(Mayforth, R. D. (1991) Ph. D. Dissertation, Univ. of Chicago). Thus, inthe absence of proliferative stimuli, resting T cells do not demonstrateexpression of the constitutively active reporter plasmid RSV-CAT.

To demonstrate that these cells could express RSV-CAT, another aliquotof the same cells was stimulated with αCD3/αCD28 and passaged inculture. 5 days later, these cells were either stimulated with PDBU/IONOor were treated with conditioned medium alone for hours, transfectedwith RSV-CAT, then harvested 40 hours later for CAT activity. Acomparison of the relative CAT activities of cells transfected either onDay 1 (resting cells) or Day 6 (proliferating cells) in the presence orabsence of PDBU/IONO prestimulation is shown in FIG. 11. PDBU/IONOprestimulation of proliferating cells resulted in a 13.4-fold increasein CAT activity relative to the proliferating non-stimulated control anda 23.7-fold increase over PDBU/IONO stimulated resting cells. Thus,cellular proliferation at the time of transfection greatly enhancesRSV-CAT reporter expression. Taken together with the results of thetransfections above, these data indicate that proliferation is requiredbut not sufficient for transgene expression.

Example 5 Differences in Reporter Gene Activity are not due toDifferences in Transfection Efficiency

As demonstrated above, TCR-dependent signal transduction prior to thetime of transfection is required for transgene expression. Stimulationafter transfection does not appreciably increase reporter activity inmost cases. The critical factor is the activational state of the cell,independent of proliferation, at the time of transfection. Any of anumber of roles for signal transduction can be envisioned. In thesimplest model, signal transduction of proliferating cells at the timeof transfection could increase transfection efficiency and therefore theamount of reporter plasmid reaching the nucleus. Alternatively, signaltransduction could facilitate the movement of transfected DNA from anon-transcribable to transcribable compartment, e.g., from the cytoplasmto nucleus. Both scenarios result in equivalent outcomes, an increasedamount of reporter plasmid in the nucleus following signal transduction.To test these possibilities, the kinetics of DNA entry and localizationwere examined by rescuing and quantitating transfected DNA from nuclearand cytoplasmic compartments at various timepoints after transfection.

Proliferating primary T cells on Day 5 were prestimulated with PDBU/IONOor treated with conditioned medium alone, transfected with the RSV-CATreporter plasmid, then cultured in complete medium. At 0, 6, 24, and 48hours after transfection, cell number and viability were determined byCoulter counting and trypan blue exclusion respectively.

Following separation into nuclear and cytoplasmic fractions by Douncehomogenization, DNA was extracted from both these fractions using serialammonium acetate/isopropanol precipitations following SDS solubilizationand Proteinase K digestion. The DNA isolation protocol is quantitativefor the recovery of both low and high molecular weight DNA. To estimatethe relative copy numbers of the transgene in nuclear and cytoplasmiccompartments at various timepoints after transfection, nuclear andcytoplasmic DNA from 10⁵ total cell equivalents was size-fractionated in1.0% agarose gels and transferred onto nitrocellulose as previouslydescribed (Thompson, C. B., and Neiman, P. E. (1987) Cell 48:369-378).Blots were hybridized with either an EcoRI fragment from the CAT codingregion of pRSV-CAT or an EcoRI/BamHI fragment from the CAT coding regionof pIL2CAT.

The results of the Southern blot analysis are presented in FIG. 12.Approximately 10⁵ copies of plasmid/cell were taken up within the first30 minutes of exposure to DNA/DEAE dextran complexes. This correspondsto approximately 10% of the total DNA transfected. Of this amount,approximately 90% localized to the nuclear fraction. Similar, orslightly increased, levels of transgene were present in the nuclear andcytoplasmic fractions of cells at 6, 24, and 48 hours aftertransfection. Significantly, there was no appreciable difference in theamount of DNA in either nuclear or cytoplasmic fractions betweenPDBU/IONO prestimulated and non-stimulated cells at any of thetimepoints. In sum, Southern analysis revealed no demonstrabledifference in the amount of DNA reaching the nuclear compartment betweensignal transduced and non-signal transduced cells.

To confirm these findings, primary T cells were transfected with theRSV-CAT or IL2-CAT reporter plasmids as described in FIGS. 7 and 10,respectively. Plasmid DNA was then isolated from the nuclear pelletfollowing hypotonic lysis of the cells.

As shown in FIGS. 13 and 14, at 40 hours post-transfection,approximately 10⁵ copies/cell of RSV-CAT plasmid were present in thenucleus, representing 10% of the total transfected DNA. As above, therewas no appreciable difference in the amount of DNA present in the nucleiof PDBU/IONO prestimulated cells, non-stimulated cells, or the nuclei ofcells stimulated after transfection with either PDBU/IONO, αCD3/αCD28,or conditioned medium. Of note, Southern analysis of DNA isolatedfollowing hypotonic lysis of cells revealed no evidence of plasmiddegradation 40 hours post-transfection.

Having recapitulated the findings using two different methods of DNAisolation, it was still not possible to discount the possibility thatthe recovery of plasmid DNA was not entirely quantitative. Toindependently confirm these results, PDBU/IONO prestimulated andnon-stimulated cells were transfected with ³²P-radiolabeled linearizedRSV-CAT. These cells were then separated into nuclear and cytoplasmicfractions 0, 6, 24, and 48 hours after transfection. The fractions werethen counted on a liquid scintillation counter.

The results, presented in FIG. 15, indicate that within 30 minutes aftertransfection, 15.2% of the total cpms transfected were taken up byPDBU/IONO prestimulated cells. This compared to 11.9% for non-stimulatedcells. Of these counts, 92% were recovered from the nuclear fraction inprestimulated cells and 84% in non-stimulated cells. At subsequenttimepoints, the % of total cpm recovered from the nuclear fractionincreased from 14.0% at 0 hours to 17.1% at 48 hours in PDBU/IONOprestimulated cells and from 10.0% at 0 hours to 13.9% at 48 hours innon-stimulated cells. This increase in nuclear counts corresponded to asmall decrease in the percentage of total cpm recovered in thecytoplasmic fractions of both prestimulated and non-stimulated cells,perhaps reflecting movement of DNA from the cytoplasm to nucleus.However, the small differences in percentage of total cpm recovered fromthe nuclear fractions of non-signal transduced and PDBU/IONO signaltransduced cells cannot account for the dramatic 67- to 345-foldincrease in RSV-CAT reporter gene expression between these twopopulations. Results from counting transfected radiolabeled DNA are inclose agreement with Southern blot analysis in terms of both theabsolute amount of plasmid entering the cell and the subsequentdistribution of that DNA between nuclear and cytoplasmic compartments.In summary, PDBU/IONO prestimulation of cells does not seem to increasereporter gene expression by increasing transfection efficiency or byfacilitating the movement of DNA from cytoplasm to nucleus.

Example 6 Superantigen-induced TCR Activation Alters the Nuclear Fate ofDNA Containing a Retroviral LTR

The above describe examples show that there exist a cellular mechanism,repressible by TCR-mediated signal transduction, which protectsquiescent and non-signal transduced proliferating primary T cells fromthe expression of exogenous DNA in vitro. Given the requirement ofTCR-dependent signal transduction for reporter gene expression, it wasinvestigated whether superantigen could serve as a sufficient stimulusfor enhanced transgene expression.

Ten hours before transfection, primary human T cells in exponentialgrowth were treated with conditioned medium alone, 5×10⁶ irradiatedautologous monocytes, SEA (10 ng/ml), or SEA (10 ng/ml) plus 5×10⁶irradiated autologous monocytes. Cells were transfected with RSV-CAT orHIV-1-CAT and harvested 40 hours after transfection and assayed for CATactivity.

The results of the CAT assays are shown in FIG. 16. In transfectionswith HIV-1-CAT, PDBU/IONO prestimulation resulted in a 15-fold increasein CAT activity relative to the non-stimulated proliferating control.Coincubation of T cells with 5×10⁶ irradiated autologous monocytesresulted in a small 2.3-fold increase in CAT activity. Treatment with 10ng/ml SEA resulted in a 32-fold increase in CAT activity, whilecoincubation of T cells with SEA+5×10⁶ irradiated autologous monocytesresulted in a 16-fold increase. Therefore, SEA, either alone, or inconjunction with APCs expressing MHC Class II (HLA-DR), increasesHIV-1-CAT reporter gene expression. Entirely analogous results are seenin transfections with RSV-CAT (FIG. 16). The proliferative status of SEAand SEA+MONO stimulated cells, as measured by tritiated thymidineincorporation and cell size, was not measurably different from that ofnon-stimulated proliferating cells. Thus, increased HIV-1-CAT andRSV-CAT reporter expression results from superantigen's effects onsignal transduction and not on proliferation per se.

Such a mechanism and its repression may be of great consequence in theevents associated with retroviral infection. For example, following theinfection of resting T cells by HIV-1, subsequent T-cell activation isrequired for integration of the HIV-1 genome into the host genome andproduction of infectious virus (Stevenson, M., Stanwick, T. L., Dempsey,M. P., and Lamonica, C. A. (1990) EMBO J. 9(5):1551-1560; Zack, J. A.,Arrigo, S. J., Weitsman, S. R., Go, A. S., Haislip, A., and Chen, I. S.Y. (1990) Cell 61:213-222; Bukrinsky, M. L., Stanwick, T. L., Dempsey,M. P., and Stevenson, M. (1991) Science 254:423-427). This suggests amodel in which HIV-1 persists in a non-productive extrachromosomal statein resting T cells until subsequent antigen or mitogen-induced T-cellactivation. Recently it has been reported that replication of HIV inresting cells requires tyrosine phosphorylation of the HIV-1 matrixprotein (Gallay, P., et al., (1995) Cell 80:379).

Superantigens, molecules recognized by T cells expressing specific TCRVβ gene products, bridge MHC Class II and the TCR, variously leading tocell activation, deletion, or anergy. This group of protein antigens ischaracterized by its ability to activate large numbers of peripheralblood T cells. Mammalian retroviruses may encode superantigens to blockgeneration of cellular immune reactivity or to facilitate replicationconsequent to direct cell activation. Recent reports suggest thatexpression of an HIV-1 superantigen may mediate the T-cell depletionseen in HIV-1 infection (Imberti, L., Sottini, A., Bettinardi, A.,Puoti, M., and Primi, D. (1991) Science 254:860-862; Cameron, P. U.,Freudenthal, P. S., Barker, J. M., Gezelter, S., Inaba, K., andSteinman, R. M. (1992) Science 257:383-387; Laurence, J., Hodtsev, A.S., and Posnett, D. N. (1992) Int. Conf. AIDS July 19-24;8(1):Th72;Pantaleo, G., Rebai, N., Graziosi, C., Lane, H. C., Sekaly, R. P., andFauci, A. S. (1992) Int. Conf. AIDS July 19-24;8(1):Th71). The nature ofthe signal transduction pathways induced by superantigen activation of Tcells remains a matter of controversy (Liu, H., Lampe, M. A., Iregui, M.V., and Cantor, H. (1991) Proc. Natl. Acad. Sci. USA 88(19):8705-8709;Kanner, S. B., Odum, N., Grosmaire, L., Masewicz, S., Svejgaard, A., andLedbetter, J. A. (1992) J. Immunol. 149(11):3482-3488; Oyaizu, N.,Chirmule, N., Yagura, H., Pahwa, R., Good, R. A., and Pahwa, S. (1992)Proc. Natl. Acad. Sci. USA 89(17):8035-8039). The efficacy of SEA,either alone, or in conjunction with APCS expressing MHC Class II, inincreasing either HIV-1-LTR or RSV-LTR driven reporter gene expressionin proliferating T cells indicates that superantigen engagement of theTCR may constitute the minimal signal necessary for exogenous DNA toenter an expressible nuclear compartment.

The examples show the existence of an active mechanism, repressible byTCR-mediated signal transduction, which protects quiescent andproliferating T lymphocytes from the expression of exogenous DNA. Tcells only express exogenous DNA following signal transduction prior totransfection. This finding has implications in the field of somatic cellgene therapy since cellular proliferation alone may be insufficient forefficient expression of exogenous DNA. Thus, the invention provides amethod for efficient expression of a gene introduced in a proliferatingT cell. In one embodiment of the invention, T cells are obtained from anindividual, stimulated to proliferate ex vivo, genetically transduced bythe method of the invention and readministered into the individual. Inthis particular embodiment, the T cells are contacted with an agent, ora combination of agents, which stimulates T cell receptor mediatedsignal transduction, such as an anti-CD3 antibody, a combination ofphorbol ester and ionomycin, or other agent that bypasses the T cellreceptor.

The invention also provides methods for blocking or decreasingexpression of exogenous DNA, such as viral DNA. Thus, primary T cellscontaining exogenous DNA, such as viral DNA, can be stimulated toproliferate while inhibiting viral replication by, for example,stimulating proliferation of the T cells with an agent that does notactivate the mechanism required for exogenous gene expression describedherein.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed is:
 1. A method for increasing the expression of anexogenous nucleic acid molecule comprising a gene, in T cells,comprising: contacting T cells in vitro with at least one stimulatoryagent, wherein the T cells are proliferating prior to contact with theat least one stimulatory agent, forming stimulated proliferating Tcells; and introducing an exogenous nucleic acid molecule comprising agene into the proliferating, stimulated T cells in vitro, at mostapproximately 24 hours after stimulation of said T cells, such that theexpression of the gene is increased in the T cells.
 2. The method ofclaim 1, wherein the T cells are contacted in vitro with at least oneproliferative agent which stimulates proliferation of the T cells priorto being contacted with the at least one stimulatory agent.
 3. Themethod of claim 1, wherein the T cells are primary T cells.
 4. Themethod of claim 1, wherein the at least one stimulatory agent is acombination of a first agent which provides a primary activation signalto the T cells and a second agent which provides a costimulatory signalto the T cells.
 5. The method of claim 4, wherein the first agentinteracts with the T cell receptor/CD3 complex.
 6. The method of claim5, wherein the first agent is an anti-CD3 antibody.
 7. The method ofclaim 4, wherein the first agent interacts with a CD2 molecule on the Tcells.
 8. The method of claim 4, wherein the first agent is an antigenon an antigen presenting cell.
 9. The method of claim 4, wherein thesecond agent is an anti-CD28 antibody.
 10. The method of claim 6,wherein the second agent is a stimulatory form of a natural ligand ofCD28.
 11. The method of claim 10, wherein the stimulatory form of anatural ligand of CD28 is the B lymphocyte antigen B7-1.
 12. The methodof claim 10, wherein the stimulatory form of a natural ligand of CD28 isthe B lymphocyte antigen B7-2.
 13. The method of claim 1, wherein the atleast one stimulatory agent is a combination of a phorbol ester and acalcium ionophore.
 14. The method of claim 1, wherein at least onestimulatory agent comprises a protein tyrosine kinase activator.
 15. Themethod of claim 1, wherein at least one stimulatory agent is asuper-antigen.
 16. A method for increasing the expression of anexogenous nucleic acid molecule comprising a gene, in primary T cells ofa subject, comprising obtaining T cells from the subject; contacting theT cells with at least one proliferative agent which stimulatesproliferation of the T cells, forming proliferating T cells; contactingthe proliferating T cells with at least one stimulating agent, formingstimulated proliferating T cells; introducing the exogenous nucleic acidmolecule comprising a gene into the stimulated proliferating T cellssuch that the expression of the gene is increased in the T cells. 17.The method of claim 16, wherein the T cells are further stimulated invitro to increase the number of T cells.
 18. The method of claim 1,wherein said nucleic acid molecule is introduced into said T cells,between approximately 1 and 24 hours after stimulation of said T cells.19. The method of claim 1, wherein said nucleic acid molecule isintroduced into said T cells, at most approximately 10 hours afterstimulation of said T cells.